Biosensors make use of the interaction of biological molecules (biomolecules) as a means of sensing an external environment. Biosensors can be very selective, due to the highly specific interactions between biomolecules, for example antibodies and their antigens, cytokines and their cell-surface receptors, enzymes and their substrates, or nucleic acids with themselves or other molecules. The species being sensed in the environment is referred to as the analyte. Therefore, the analyte can be another biological molecule or a chemical that interacts with a biorecognition molecule that has high selectivity for the target analyte. Further, signal transduction methods combined with amplification can provide a biosensor with high sensitivity. These properties—selectivity and sensitivity—make biosensors particularly attractive as analytical devices.
To be useful as a biosensor, the biological interaction must be transduced into a measurable signal. Typically, this interaction can be detected colorimetrically, calorimetrically, or electrochemically. Often, the biological interaction can be converted into an electrochemical signal. In such an electrochemical biosensor, the biorecognition molecule is typically immobilized in a bioselective layer in intimate contact with an electrode which measures the movement of electrons or ions exchanged in the biological interaction. Typically, voltammetry or amperometry can be used to measure the current response to a controlled applied potential that induces an oxidation or reduction process. Alternatively, potentiometry can be used to measure the voltage response to a controlled current.
Functionalization of conducting and semiconducting surfaces is a vital component in the fields of bioelectronics, molecular electronics, clinical diagnostics, and chemical and biological sensing (see I. Willner et al., Biosens. Bioelectron. 22, 1841 (2007); S. J. Oh et al., OMICS 10 327 (2006); R. Byrne and D. Diamond, Nat. Mat. 5, 421 (2006); and J. J. Gooding, Anal. Chim. Acta, 559, 137 (2006)). Of particular interest is the ability to conjugate a surface with two or more different biological, redox active, and/or photo/chemical sensitive molecules. Such multifunctional surfaces would facilitate collection of complicated data sets that would be relevant to cell signaling studies, genomic and proteomic analysis, and the proof-positive identification of biological organisms (see I. Medintz, Nat. Mat. 5, 842 (2006); R. Polsky et al., Electroanalysis 20, 671 (2008); C. A. Rowe-Taitt et al., Biosens. Bioelectron. 15, 579 (2000); C. A. Rowe et al., Anal. Chem. 71, 3846 (1999); and J. C. Harper et al., Langmuir 23, 8285 (2007)). Several methods have been devised and employed to allow surface conjugation including photolithography (see D. Falconnet et al., BioMaterials 27, 3044 (2006); E. Delamarche et al., Adv. Mater. 17, 2911 (2005); and F. L. Yap and Y. Zhang, Biosens. Bioelectron. 22, 775 (2007)), self-assembling monolayers (SAMs) (see E. Katz and I. Willner, Angew. Chem., Int. Ed. 43, 6042 (2004); J. J. Gooding et al., Electroanalysis 15, 81 (2003); and Y. Xiao et al., Science 299, 1877 (2003)), silanes (see W. Senaratne et al., Biomacromolecules 6 2427 (2005); and N. K. Chaki et al., Biosens. Bioelectron. 17, 1 (2002)), stamping (see R. S. Kane et al., Biomaterials 20, 2363 (1999); and Y. Xia and G. M. Whitesides, Angew. Chem., Int. Ed. 37, 550 (1998)), mechanical and/or electrochemical removal of material (see J.-W. Jang et al., Nano Lett. 8, 1451 (2008); G. Liu et al., Acc. Chem. Res. 33, 457 (2000); J. K. Schoer and R. M. Crooks, Langmuir 13, 2323 (1997); and J. K. Schoer et al., J. Phys. Chem. 100, 11086 (1996)), and electropolymerization (see E. Stern et al., Anal. Chem. 78, 6340 (2006); K. Kim et al., J. Chem. Commun., 4723 (2006); and S. Cosnier, Anal. Bioanal. Chem. 377, 507 (2003)). Of these techniques, SAMs based on alkanethiol-gold surfaces have been the most widely used. Several studies utilizing SAMs to form mixed surfaces demonstrating multifunctionality have been published (see S. Choi and W. L. Murphy, Langmuir 24, 6873 (2008); B. M. Lamb et al., Langmuir 24, 8885 (2008); C. Boozer et al., Sens. Actuators, B Chem. 90 22 (2003); R. G. Chapman et al., Langmuir 16, 6927 (2000); C. Roberts et al., J. Am. Chem. Soc. 120, 6548 (1998); and N. Patel et al., Langmuir 13, 6485 (1997)). However, the low enthalpy of the Au—S bond, mobility of the SAMs on gold, tendency of the Au—S bond to oxidize in air and media, and low potential stability window have limited the usefulness of this chemistry (see T. M. Willey et al., Surf. Sci. 576, 188 (2005); and N. T. Flynn et al., Langmuir 19, 10909 (2003).
Electrode surface modification by the electrochemical reduction of aryl diazonium salts is a promising alternative to conventional electrode modification schemes (see M. Delamar et al., J. Am. Chem. Soc. 114, 5883 (1992); P. Allongue et al., J. Am. Chem. Soc. 119, 201 (1997); A. J. Downard, Electroanalysis 12, 1085 (2000); M.-C. Bernard et al., Chem. Mater. 15, 3450 (2003); T.-C. Kuo et al., Electrochem. Solid St. 2, 288 (1999); and F. Anariba et al., Anal. Chem. 75, 3837 (2003)). Electroreduction of the diazonium produces an aryl radical that can then graft to a conducting or semiconducting surface forming a stable covalent bond. This approach has several advantages over alkanethiol-gold chemistry including ease of surface modification, a wider potential window for subsequent electrochemistry, and high stability under long term storage in air and during potential cycling under acidic conditions (see G. Liu et al., J. Electroanal. Chem. 600, 335 (2006); and G. Liu et al., Chem. Phys. 319, 136 (2005)). Diazonium salts with a wide range of substituent groups useful for surface functionalization have been reported including biotin (see M. Dequaire et al., J. Am. Chem. Soc. 121, 6946 (1999), maleimide (see J. C. Harper et al., Langmuir 24, 2206 (2008)), carboxyl (see B. P. Corgier et al., J. Am. Chem. Soc. 127, 18328 (2005); R. Polsky et al., Biosens. Bioelectron. 23 757 (2008); and R. Polsky et al., Lanmuir 23, 364 (2007)), amine (see C. S. Lee et al., Nano Lett. 4, 1713 (2004); A. Ruffien et al., Chem. Commun., 912 (2003); and A. Shabani et al., Talanta 70, 615 (2006)), thiol (see L. T. Nielsen et al., J. Amer. Chem. Soc. 129, 1888 (2007)), boronic acid (see R. Polsky et al., Angew. Chem., Int. Ed. 47, 2631 (2008)), and azide or alkyne for click chemistry (see D. Evrar et al., Chem. Eur. J. (2008), DOI: 10.1002/chem.200801168). Another significant advantage of diazonium electrodeposition chemistry over alkanethiol surfaces is that the surface coverage and density of the resulting film can be controlled by the experimental conditions yielding submonolayer to multilayer films (see F. Anariba et al., Anal. Chem. 75, 3837 (2003); and P. A. Brooksby and A. J. Downard, Langmuir 20, 5038 (2004)). However, it may not always be synthetically straightforward to incorporate the various substituents into a single molecule.
Previously, two-component films prepared from aryl diazonium salts have been formed by simultaneous assembly of two diazonium compounds in a single solution resulting in mixed surfaces (see G. Liu et al., Chem. Phys. 319, 136 (2005); G. Liu and J. J. Gooding, Langmuir 22, 7421 (2006); and C. Louault et al., ChemPhysChem 9, 1164 (2008)) or consecutive deposition leading to stacked structures (see A. Adenier et al., Chem. Mater. 18, 2021 (2006); P. A. Brooksby and A. J. Downard, J. Phys. Chem. B 109, 8791 (2005); P. A. Brooksby and A. J. Downard, Langmuir 21, 1672 (2005); and A. O. Solak et al., Anal. Chem. 75 296 (2003)). Poly(dimethylsiloxane) PDMS molds have also been used to pattern assembly of two differing diazoniums via fill-in or consecutive assembly (see A. J. Downard et al., Langmuir 22, 10739 (2006)). These works employ electrochemistry, XPS, AFM and other techniques to elucidate the chemical composition of the resulting binary films and the dependence of film properties on the deposition conditions. To date, only one study has utilized diazonium electrodeposition for formation of a multifunctional film towards a specific application. In this work, Liu and Gooding prepared a two-component carbon surface by electroreduction of a mixture containing diazoniums with oligo(phenylethynlene) and poly(ethylene glycol) (PEG) functionality (see G. Liu and J. J. Gooding, Langmuir 22, 7421 (2006)). Oligo(phenylethynlene) served as a conductive path to the electrode allowing direct electron transfer to surface immobilized horseradish peroxidase or myoglobin, while PEG served to decrease non-specific adsorption of bovine serum albumin and components of blood serum onto the electrode surface.
However, a need remains for more versatile and simple methods to prepare multifunctional thin films using aryl-onium chemistry.